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MCB 502A-2014. Lecture #14.Chromosomal damage and itsRecombinational Repair— Chromosome cycle-dependent chromosomal lesions— Classification of chromosomal lesions: types of "failure-by-design"— Chromosomal lesions are repaired by Rec-dependent processes— Homologous recombination: exchange by resolution of Holliday junctions— Genetics of Homologous Recombination— The principles of repair of chromosomal lesions— Biochemistry of recombinational repair— Major types of chromosomal lesions and corresponding recombinational repairpathways— Daughter-strand gap repair— When recombinational repair of daughter-strand gaps is impossible— Double-strand end repair— Replication restart— Chromosomal dimer resolution— When recombinational repair of double-strand breaks is unavailable— Recombinational misrepair: the importance of sister-chromatid cohesionChromosome cycle-dependentchromosomal lesions-1— Double-strand breaks and interstrandcrosslinks are classified as direct chromosomallesions, because they do not require any otherendogenous (cellular) processes for theirformation.— However, they do assume that cellsencounter unusual exogenous interference inthe form of high-energy ionizing radiation orcross-linking agents.— For this reason, direct chromosomal lesionsrepresent a small minority of all chromosomallesions that could form in growing cell.— The bulk of chromosome lesions in cyclingcells are due to transitions of the chromosomecycle on non-intact DNA.DSB65°C95°CChromosome cycle-dependentchromosomal lesions-2— Replication-dependent chromosomallesions are induced when replication forksrun into unrepaired one-strand lesions intemplate DNA.— For example, replicating DNA in uvrmutant cells, which were irradiated with UV,is analyzed in sucrose gradients, which candistinguish chromosome-size DNA piecesfrom smaller pieces.— Pyrimidine dimers in uvr mutant cells arenot removed, and so replication forks in UV-irradiated uvrA mutants will encounter PDson the regular basis.T=TChromosome cycle-dependentchromosomal lesions-3— If one runs pre-labeled (labeled beforeUV) chromosomal DNA from these cellsthrough alkaline sucrose gradient, one willsee no change from unirradiated control cells.— However, if one now labels DNA in thesecells for 10 minutes after UV irradiation, sothat only the newly-replicated DNA islabeled, one will see that the label inunirradiated cells accumulates in HMWspecies, as first described by Okazaki(because the labeling time is so long), but inthe irradiated cells, the label accumulates inlower-molecular weight species, and theheavier the irradiation, the lower themolecular weight of the newly-synthesizedDNA becomes.3H-DNABottom(HMW)Top(LMW)Neutral (dsDNA)No UVAfter UVBottom(HMW)Top(LMW)Alkaline (ssDNA)No UVAfter UVLabeled just before UV irradiation3H-DNABottom(HMW)Top(LMW)Neutral (dsDNA)No UVAfter UVBottom(HMW)Top(LMW)Alkaline (ssDNA)No UVAfter 1xUVAfter 3xUVLabeled right after UV irradiationChromosome cycle-dependentchromosomal lesions-3— This result suggests that, when replicatingDNA strands encounter PDs, they have to goaround them, leaving behind single-strandinterruptions, whose number roughly correspondsto the number of the original PDs, encounteredby the replication forks.T=TT=TT=TChromosome cycle-dependentchromosomal lesions-4— Actually, these are not just interruptions or evenshort gaps opposite PDs — the size of the gaps isapproximately half the length of Okazaki fragments— from 500 to 1,000 nucleotides.— They are called daughter-strand gaps, becausethey are generated only in the newly-synthesizedDNA strands.— It is proposed that when a replication forkencounters an unrepaired pyrimidine dimer, theDNA polymerase copying the affected strand isstalled, but the replication fork continues past thelesion, recruiting another polymerase andreinitiating downstream.— Such a maneuver leaves a portion of the newly-synthesized strand unreplicated, — thus, adaughter-strand gap.T=T1/2 OkazakifragmentT=TAnotherpolymeraserecruitedPolymerasestalled at PDChromosome cycle-dependentchromosomal lesions-5— The daughter-strand gap is achromosomal lesion, because itprecludes subsequent replication ofthe affected chromosome.— Indeed, both strands of the duplexwith a daughter-strand gap nowcannot serve as templates forsubsequent replication: one has thegap, the other still has the original PD.— When a daughter-strand gaphappens to form in excision-repairproficient cells, there is yet anotherunpleasant outcome: since the PD isnow in a single-strand DNA, it cannotbe excised by NER, which works onlyin duplex DNA.T=TInterruptedby the gapBlocked byPDPD in ssDNAis resistant toNERReplication amplifies DNAdamageOne-strandDNA lesionReplicationTwo-strandDNA lesion(Chromosomallesion)— A new concept: replication duplicates not only DNA, but also DNAdamage.— In addition, duplicated DNA damage takes on a new quality, becausenow it blocks the chromosome cycle.— Real life lesson: do not use something half-broken — you may ruin itbeyond repair.Do not use something half-broken— you may ruin it beyond repair(…besides thatit is sometimesjust crazydangerous!)Chromosome cycle-dependentchromosomal lesions-6— If we analyze a replicatingchromosome from UV-irradiated WTcells (nucleotide-excision repair isoperational in this case), we will see,in contrast to the uvr mutant cells, thatthe molecular weight of the pulse-labeled DNA strands in alkalinesucrose gradients approaches the oneof the intact chromosomal DNA,suggesting a greatly reducedformation of daughter-strand gaps.— This is expected, because in WTcells, PDs are rapidly removed bynucleotide-excision repair.3H-DNABottom(HMW)Top(LMW)Neutral (dsDNA)No UVAfter UVBottom(HMW)No UVAfter UVLabeled just before UV irradiation3H-DNABottom(HMW)Top(LMW)Neutral (dsDNA)No UVAfter UVBottom(HMW)Labeled right after UV irradiationNo UVAfter 1xUVAfter 3xUVTop(LMW)Alkaline (ssDNA)Top(LMW)Alkaline (ssDNA)Chromosome cycle-dependentchromosomal lesions-7— However, if we run a control, bytesting the overall chromosome status inneutral sucrose gradients, then we willsee that the MW of duplex DNA isactually decreased, suggesting that thechromosome fragments (forms occasionaldouble-strand breaks) in these conditions.— If true, these breaks should also affectthe old DNA strands (labeled before UV-irradiation).— In fact, they do: a fraction of the oldDNA strands (expected to be in duplexwith strands
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